The use of labeled molecules as probes for the detection of other molecules, such as DNA or other materials, is as important activity from a commercial, diagnostic and scientific point of view. Historically, the labels utilized for such purposes have been radioactively labeled with isotopes of hydrogen (.sup.3 H), phosphorous (.sup.32 P), carbon (.sup.14 C), or iodine (.sup.125 I). Such radioactive compounds provide useful probes that permit the user to detect, monitor, localize or isolate the labeled molecule of interest. To date, radioactive materials have provided the most sensitive, and in many cases, the only means to perform many important experimental or analytical tests. There are, however, serious limitations and drawbacks associated with the use of radioactive compounds. First, since personnel who handle radioactive material can be exposed to potentially hazardous levels of radiation, elaborate safety precautions must be maintained during the preparation, utilization, and disposal of the radioisotopes. Second, radioactive labeled molecules are extremely expensive to purchase and use, in large part due to the cost of equipment and manpower necessary to provide the appropriate safeguards, producer/user health monitoring services and waste disposal programs. Third, radioactive materials are often very unstable and have a limited shelf life, which further increases usage costs. This instability results from radiolytic decomposition due to the destructive effects associated with the decay of the radioisotope itself and from the fact that many isotopes have half lives of only a few days.
Recently it has been discovered that chemically labeled probes can be utilized in place of radioactively labeled probes. For example, probes consisting of nucleotides labeled with biotin that are capable of being incorporated into double stranded DNA have been prepared. The single stranded DNA labeled with biotin is capable of detecting its target, a complementary single stranded DNA, by hybridizing with such complementary single stranded DNA. The presence of the biotinylated nucleotide in the hybridized double stranded DNA is detected by utilizing the strong affinity of avidin for biotin which results in the formation of a stable biotin/avidin complex. The binding constant for the biotin/avidin complex is about 10.sup.-16. Also attached to the avidin, which has four binding sites for biotin, is a biotinylated enzyme, such as biotinylated horseradish peroxidase, which is capable of generating a signal when treated with a suitable reagent. Streptavidin can be used as a substitute for avidin. The utilization of streptavidin or avidin for the detection of biotinylated nucleotides or biotinylated DNA is disclosed in European Patent Application Publication No. 0,063,879, published March 11, 1983. This European Patent Application Publication is derived from U.S. application Ser. No. 225,223 filed April 17, 1981. The disclosures of the above referred European Patent Application Publication and the U.S. Application are herein incorporated and made part of this disclosure.
It has also been proposed to label nucleotides with glycosyl groups, such as maltose, lactose, mannose, maltose triose and mannose triose. Such labeled nucleotide can be recognized with a lectin, such as Concanavalin A. Lectins possess an affinity for glycosyl groups, albeit the affinity of a lectin for a glycosyl group is not nearly as strong as the affinity of avidin for biotin. The binding constant is about 10.sup.-4. Thus, a glycosyl group/lectin complex is formed. Attached to a lectin, which has four binding sites for a glycosyl group, is a glycosylated enzyme, such as alkaline phosphatase or acid phosphatase. The glycosylated enzyme is capable of creating a signal when treated with a suitable reagent. This detection system is disclosed in co-pending co-assigned U.S. patent application Ser. No. 391,440 filed June 23, 1982 now abandoned. The disclosures of this U.S. Patent Application are herein incorporated and made part of this disclosure.
Each of the above detection systems is a homologous detection system. A homologous detection system contains only one kind of complex forming site, i.e., biotin/avidin or glycosyl group/lectin. The same affinity is utilized to (1) recognize the label of the probe and (2) connect a moiety that is capable of generating a signal to the detection system. Thus, for example, the affinity between avidin and biotin is utilized to recognize the biotinylated probe and to connect a biotinylated enzyme to avidin, the enzyme being capable of generating a signal when treated with a suitable reagent.